THE USDA ANIMAL GENOME BLUEPRINT TARGET AREAS INCLUDE A FOCUS ON CHARACTERIZATION OF GENOME FUNCTIONAL ELEMENTS IN HIGH THROUGHPUT ASSAYS ACROSS MULTIPLE INDIVIDUALS AND BREEDS. THE NEXT MAJOR CHALLENGE FOR FUNCTIONAL ANNOTATION OF ANIMAL GENOMES (FAANG) IS TO LINK GENOMIC VARIATION IN REGULATORY SITES TO DIFFERENCES IN GENE EXPRESSION AND PHENOTYPE. THE FOCUS OF FAANG ON DEEP CHARACTERIZATION OF A FEW ANIMALS IMPLICITLY ASSUMES THOSE ANIMALS ARE REPRESENTATIVE OF THE WHOLE POPULATION. FOR MANY OF THE DATA TYPES AND TISSUES STUDIED WITHIN FAANG, THERE IS LIMITED UNDERSTANDING OF THE VARIABILITY WITHIN POPULATIONS, SUBSPECIES, OR BREEDS. WE WILL ADDRESS THIS LIMITATION OF THE FAANG APPROACH BY APPLYING CAGE-SEQ IN MANY ANIMALS. OUR PROJECT WILL (1) IDENTIFY ENHANCERS FROM WHOLE BLOOD FOR THE TWO BOVINE SUBSPECIES, AND (2) QUANTIFY THE EFFECT OF BOVINE ENHANCER VARIATION ON GENE EXPRESSION. FOR OBJECTIVE 1, WE WILL TEST THE HYPOTHESIS THAT ENHANCERS DIFFER BETWEEN AND WITHIN THE SUBSPECIES OF CATTLE. FOR OBJECTIVE 2, WE WILL TEST THE HYPOTHESIS THAT VARIATION IN ACTIVITY OF ENHANCERS ALTERS EXPRESSION OF THE GENES THEY REGULATE IN WHOLE BLOOD. AS ONE APPLICATION OF THE RESULTANT ATLAS OF ENHANCER VARIATION, WE WILL DETERMINE THE EXTENT TO WHICH VARIATION WITHIN ACTIVE ENHANCERS CONTRIBUTES TO PHENOTYPIC VARIANCE, AND THE EXPRESSION OF COMPLEX, POLYGENIC TRAITS. THE EFFECT OF ENHANCER VARIATION ON GENE EXPRESSION WILL BE STATISTICALLY VALIDATED IN AN UNRELATED POPULATION AND USING CRISPR/CAS MEDIATED GENE EDITING TO EXCHANGE ENHANCER ELEMENTS IN CULTURED BOVINE CELLS. LEVERAGING OUR UNIQUE ANIMAL RESOURCES, WE ANTICIPATE WE WILL CAPTURE A SIGNIFICANT PROPORTION OF VARIABLE ENHANCER SITES EXPECTED TO EXIST IN BLOOD WITHIN THE US BEEF HERD. OUR RESEARCH IS SIGNIFICANT BECAUSE IT WILL CONTRIBUTE TO UNDERSTANDING THE WAY IN WHICH GENE REGULATION OCCURS AT ENHANCER SITES IN THE TWO BOVINE SUBSPECIES.